Mechanisms of Migraine Migraine is a debilitating episodic pain disorder for which there are no consistently effective therapeutic interventions. It is also one of the most prevalent pain disorders afflicting as many as 10% of the general adult population and 18% of women. Identification of novel approaches for the treatment of migraine is therefore highly significant. The prevailing weight of evidence indicates that the primary afferent neurons innervating the dura and dural vasculature are the source of the pain of a migraine attack. The present proposal is therefore focused on components of the dura that can influence afferent activity. These components include resident and recruited immune cells in the dura and the dural vasculature. We will also study the afferents themselves. We have proposed to exploit two unique features of migraine as a means to identify mechanisms that enable the initiation of a migraine attack. One is that stress is the most common trigger of a migraine attack. A second is that migraine attacks occur during relaxation phase after stress has ended. We propose that sympathetic post-ganglionic neurons (SPGN) in the dura serve as a link between stress and migraine because they are a critical component of the stress response system, the dura is heavily innervated by SPGN terminals, and all three dural components to be studied are regulated by mediators released from SPGN terminals. Finally, we also propose that sex is a critical factor that influences the link between stress and migraine because of the higher prevalence of migraine in women and the fact that gonadal hormones, in particular estrogens co- regulate each of the dural components to be studied. Thus, the central hypothesis of this proposal is that that stress drives sex- and SPGN-dependent changes in the regulation of dural immune cells, vasculature and primary afferents, that set the stage for the initiation of a migraine attack. This hypothesis will be tested in experiments described under three specific aims. In the first, we will determine the impact of sex, SPGN innervation, and persistent stress on resident and recruited immune cells in the dura. In the second, we will determine the impact of sex and persistent stress on SPGN-dependent regulation of the dural vasculature. In the third, we will determine the impact of sex and persistent stress on SPGN-dependent changes in voltage-gated Ca2+ currents in dural afferents and dural afferent excitability. The proposed experiments will not only provide valuable insight into the neurobiology of the dura, a structure critical for the health of the brain, but suggest novel approaches for the treatment of migraine enabling the prevention an attack altogether.